The gene regulatory network that controls pluripotency and differentiation in embryonic stem cells (ESCs) is widely studied and becoming well understood. However, the genetic parameters of successful somatic cell nuclear transfer and the genetic program that sets up the ESC gene network are not known. The answers to these key questions in ESC research lie in the gene regulation of the early embryo, which for our purpose here, encompasses developmental stages that occur after fertilization and prior to blastocyst formation. Just as the inner cell mass of the blastocyst must maintain pluripotency and the ability to self-renew in order to give rise to cell lineages that form the fetus, the blastomeres of the early embryo must establish totipotency and self-renewal abilities to form the blastocyst. However, we do not know how totipotency of blastomeres is achieved and how it relates to the waves of embryonic genome activation. Therefore, our overall goal is to address both the general architecture of this gene network and identify specific regulators that are critical and sufficient for establishing the correct genetic circuitry at the 1- to 2-cell stages, and that would ensure subsequent developmental competence. Further, the function of these regulators will be determined in order to understand mechanisms that are essential in early embryo development. We have established experimental strategies to interrogate the precise roles of transcriptional regulators during the maternal-embryonic transition of the early embryo, when both maternal and early embryonic transcripts may be present simultaneously. We discovered that Oct4, a homeodomain transcription factor of the POU family that is known for its critical functions in pluripotency in the inner cell mass, ESCs, and germ cells, has a novel role in early embryo development prior to the blastocyst stage and is required for progression beyond the multi-cell and morula stages. In addition, our data suggest that the pluripotency regulators, Sox2 and Sall4, may also have critical functions prior to blastocyst formation. We propose to fully investigate the novel roles of these and other pluripotency regulators and use them as portals to dissect gene regulation in the early embryo in the context of nuclear reprogramming and embryonic genome activation. Understanding the genetic requirements of the early embryo will have a direct and significant impact on regenerative and stem cell medicine, the treatment of infertility, and cancer research, as genes implicated in cancer are highly enriched in this genetic program. Public Health Relevance: The overall goal of this project is to investigate the novel roles of Oct4, Sall4, Sox2, and other pluripotency regulators in the early mouse embryo, with a focus on the 1- to 2-cell stages during the maternal-embryonic transition. Using these transcriptional regulators as portals, we will dissect the gene regulatory network in the early embryo and identify specific regulators that are critical and sufficient for ensuring subsequent developmental competence. Understanding the genetic requirements of the early embryo will have a direct and significant impact on regenerative and stem cell medicine, the treatment of infertility, and cancer research.